Sugar chain synthase gene

ABSTRACT

The present invention provides a method effective for diagnosis or treatment of congenital disorders of glycosylation syndrome (CDGS) by clarifying the gene of the N-linked sugar chain synthase in human endoplasmic reticulum. In the present invention, a gene of an enzyme catalyzing human N-linked sugar chain synthesis is found based on, as indicators, whether it is homologous with the gene of the enzyme catalyzing N-linked sugar chain synthesis in yeast endoplasmic reticulum and compliments the function of the gene for a deletion yeast strain of the gene.

TECHNICAL FIELD

The present invention relates to a human gene for synthesizing ahuman-derived N-linked sugar chain; an agent for diagnosing or treatingcongenital disorders of glycosylation syndrome (CDGS) by using the gene;a recombinant vector and a transformant which are integrated with thegene; a process for producing an enzyme catalyzing a human N-linkedsugar chain synthesis by using the transformant; or a method forsynthesizing a human N-linked sugar chain by using the enzyme or thetransformant.

BACKGROUND ART

In order to identify a causative gene of congenital disorders ofglycosylation syndrome (CDGS), it is required that genes relating tosugar chain synthesis should comprehensively be cloned. Particularly,genes in the synthetic processes in human endoplasmic reticulum whichrelate to the essential synthesis of a human N-linked sugar chain areparticularly important.

Actually, some causative genes of congenital disorders of glycosylationsyndrome have been investigated. However, it is known that the syndromeis mostly caused by genes relating to the synthesis of an N-linked sugarchain in endoplasmic reticulum. The synthetic pathway of an N-linkedsugar chain in endoplasmic reticulum is conserved in yeast up to humans.Most of the genes relating to the synthesis have been isolated fromyeast.

Although it is considered that almost all sequences of human genes existon database, most of the genes have not yet been isolated because thefunctions are unknown. Thus, isolation of these genes is an importantproblem for further detailed diagnosis and treatment of CDGS.

On the other hand, the enzymes for the fundamental biosynthesis inendoplastic reticulum for the synthesis of an N-linked sugar chain areessential for the synthesis in vitro at a large scale. Therefore, theisolation of these genes is very important for the supply of the enzymesas an application to sugar chain engineering.

According to the present invention, the human gene relating to theN-linked sugar chain synthesis in endoplasmic reticulum is clarified,congenital disorders of glycosylation syndrome is diagnosed and treatedby using it, and a method for synthesizing the enzyme at a large scaleis provided as an application to sugar chain engineering.

DISCLOSURE OF THE INVENTION

The inventors made intensive studies in order to solve the problems, andfound a human gene which is highly homologous with an enzyme catalyzingN-linked sugar chain synthesis in yeast endoplasmic reticulum and thencloned it. Surprisingly, the cloned human gene complimented the functionof the gene for a deletion strain of the gene in yeast endoplasticreticulum. Thus, the inventors were convinced that the human gene wouldbe a gene of an N-linked sugar chain synthase in human endoplasmicreticulum. Thus, the present invention has been achieved.

The present invention relates to the followings.

-   (1) A human gene for synthesizing an enzyme catalyzing human    N-linked sugar chain synthesis, which is homologous with a gene of    an enzyme catalyzing N-linked sugar chain synthesis in yeast    endoplasmic reticulum, and is capable of complimenting the function    of said gene for a deletion yeast strain of said gene.-   (2) The human gene according to the above-described (1), wherein the    enzyme catalyzing human N-linked sugar chain synthesis is a    glycosyltransferase.-   (3) A gene which encodes the amino acid sequence represented by SEQ    ID NO:2, 4, 6, 8 or 10 or a protein which comprises an amino acid    sequence in which one or more amino acids in the amino acid sequence    represented by SEQ ID NO:2, 4, 6, 8 or 10 are deleted, substituted    or added.-   (4) A gene which comprises the nucleotide sequence represented by    SEQ ID NO:1, 3, 5, 7 or 9.-   (5) An agent for diagnosing or treating human congenital disorders    of glycosylation syndrome, which comprises using the gene encoding    the amino acid sequence according to the above-described (3) or the    gene represented by SEQ ID NO: 1, 3, 5, 7 or 9.-   (6) A recombinant vector which is integrated with the gene according    to any one of the above-described (1) to (3).-   (7) A transformant which is transformed by the recombinant vector    according to the above-described (6).-   (8) A process for producing an enzyme catalyzing human N-linked    sugar chain synthesis, which comprises culturing the transformant    according to the above-described-   (7) in a culture, and collecting the enzyme catalyzing human    N-linked sugar chain synthesis from the culture.-   (9) A method for synthesizing a human N-linked sugar chain, which    comprises using the enzyme according to the above-described (8).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of electrophoresis of a transformant sample, asample of JY746 strain (wild type strain) and a sample of gmd3 strain.

FIG. 2 shows results of electrophoresis of a transformant sample, asample of W303-1A strain (wild type strain) and a sample of alg8 strain.

FIG. 3 shows results of electrophoresis of a transformant sample, asample of W303-1A strain (wild type strain) and a sample of alg9 strain.

FIG. 4 shows results of electrophoresis of a transformant sample, asample of W303-1A strain (wild type strain) and a sample of alg10strain.

FIG. 5 shows results of electrophoresis of a transformant sample, asample of W303-1A strain (wild type strain) and a sample of alg12strain.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, genes for use in cloning gene of an enzymecatalyzing human N-linked sugar chain synthesis are genes of an enzymegroup relating to N-linked sugar chain synthesis in yeast endoplasmicreticulum, such as genes of ALG11 gene, ALG8 gene, ALG9 gene, ALG10 geneand ALG12 gene. Examples include alg11 gene of Schizosaccharomycespombe, ALG8 gene, ALG9 gene, ALG10 gene and ALG12 gene of Saccharomycescerevisiae, and the like.

The alg11 gene of Schizosaccharomyces pombe is a gene encodingglycolipid α-mannosyltransferase (EC 2.4.1.131) in the N-linked sugarchain synthesis system.

The ALG8 gene of Saccharomyces cerevisiae is a gene encoding glycolipidα-glycosyltransferases (EC 2.4.1._) in the N-linked sugar chainsynthesis system.

The ALG9 gene of Saccharomyces cerevisiae is a gene encoding glycolipidα-mannosyltransferase (EC 2.4.1.130) in the N-linked sugar chainsynthesis system.

The ALG10 gene of Saccharomyces cerevisiae is a gene encoding glycolipidα-glucosyltransferases (EC 2.4.1._) in the N-linked sugar chainsynthesis system.

The ALG12 gene of Saccharomyces cerevisiae is a gene encoding glycolipidα-mannosyltransferase (EC 2.4.1.130) in the N-linked sugar chainsynthesis system.

A human gene which is homologous with these yeast genes and is capableof complimenting the function for a deletion or mutation yeast strain ofthese genes is a gene of an enzyme in the N-linked sugar chain synthesissystem in human endoplasmic reticulum.

In order to obtain the gene of the enzyme in the N-linked sugar chainsynthesis system in human endoplasmic reticulum in the presentinvention, a human gene which is homologous with a gene of an enzyme inthe N-linked sugar chain synthesis system in yeast endoplasmic reticulumis cloned. In this cloning, human cloned DNA which is homologous withthe gene of the enzyme in the N-linked sugar chain synthesis system inyeast endoplasmic reticulum can be obtained, for example, by preparingsynthetic primers based on the nucleotide sequence of the gene of theenzyme in the N-linked sugar chain synthesis system in yeast andcarrying out PCR using a human cDNA library.

Then, the human gene which is homologous with the gene of the enzyme inthe N-linked sugar chain synthesis system in yeast endoplasmic reticulumis ligated to a vector which can be expressed in yeast, such as pREP1,YEp51, YEp352GAP, pSH19, and pYO325, to transform yeast in which thegene of the enzyme in the N-linked sugar chain synthesis system isdeleted or mutated with the recombinant expression vector. When thetransformed yeast recovers the function lost by the deletion or mutationof the gene of the enzyme, the human gene is considered to be the geneof the enzyme in the N-linked sugar chain synthesis system in humanendoplasmic reticulum. Then, a large number of the recombinant vectorsare collected by PCR amplification or culturing of the transformant, andthe gene of the enzyme in the N-linked sugar chain synthesis system inhuman endoplasmic reticulum can be obtained by known methods in thefield, such as cleavage of the vector with restriction enzymes.

In this point, more specifically, for example, the alg11 gene ofSchizosaccharomyces pombe is a gene encoding glycolipidα-mannosyltransferase (EC2.4.1.131) in the N-linked sugar chainsynthesis system, and the gmd3 strain of Schizosaccharomyces pombe inwhich this gene is mutated is temperature-sensitive and is deficient insugar chain addition, so that it produces an acidic phosphatase having amolecular weight smaller than that of the wild type strain because sugarchain addition of the acidic phosphatase which is a glycoprotein isdeficient. In this connection, a human gene (for example, FLJ21803)which is highly homologous with the alg11 gene is obtained by preparingprimers based on the sequence of the alg11 gene and carrying outamplification by PCR using a human cDNA library. Using the gene, thegmd3 strain is transformed to thereby examine the temperaturesensitivity and the molecular weight of the acidic phosphatase. When thetransformant is negative with the temperature sensitivity and themolecular weight of the acidic phosphatase is returned to the same levelas that of the wild type strain, the human gene is the gene of theenzyme in the N-linked sugar chain synthesis in human endoplasmicreticulum and can be produced in a large amount according to the usualmethod.

The yeast strain in which the gene of the enzyme in the N-linked sugarchain synthesis system is deleted or mutated includesSchizosaccharomyces pombe gmd3 strain in which the alg11 gene ismutated, Saccharomyces cerevisiae alg8 strain in which the ALG8 gene ismutated, and the like. The yeast strain can be obtained by mutatingyeast according to a mutagenic method, such as radiation and ultravioletirradiation, and screening a yeast strain in which the gene of theenzyme in the N-linked sugar chain synthesis is deleted or mutated byusing decrease of the molecular weight of the glycoprotein or thetemperature sensitivity as an indicator.

CDGS is an autosomal recessive genetic disease and causes variousdisorders such as cerebellar hypoplasia, liver disorders and peripheralnerve disorders. Among these, it is considered that type I CDGS iscaused by the deficiency of the enzyme due to the deletion or mutationof the gene of the enzyme in the N-linked sugar chain synthesis. Thegene identified as the gene of the enzyme in the human N-linked sugarchain synthesis system for the first time in the present invention is auseful diagnostic agent of CDGS. Whether or not a patient suffers fromCDGS can be diagnosed by comparing the nucleotide sequence of glycolipidα-mannosyltransferase (EC 2.4.1.131) represented by SEQ ID NO:1 with thenucleotide sequence of the corresponding gene of the enzyme of thepatient to detect abnormality of the gene.

When the diagnosis is carried out by using the gene of the enzyme in thehuman N-linked sugar chain synthesis system in the present invention,the gene of the enzyme of the patient as a comparative subject can beobtained by collecting the subject gene from patient blood using thegene of the enzyme of the present invention as probe and amplifying itappropriately by PCR.

The gene of the enzyme in the human N-linked sugar chain synthesissystem in the present invention is also useful for gene therapy. For thetherapy, the gene of the enzyme in the human N-linked sugar chainsynthesis system in the present invention is inserted into a vector forgene therapy, such as adenovirus, retrovirus or Sendai virus, to prepareviral particles containing the gene using helper cells or the like, andthe viral particles are inoculated into human bodies to introduce thegene of the enzyme.

In the present invention, the gene of the enzyme in the human N-linkedsugar chain synthesis system is inserted into a vector, such as plasmidpBR322, pUC18, pUC19, pET-3, YEp51 or YEp352GP, to transform a host,such as bacterium or yeast, with the vector. The resulting transformantis cultured in a culture to prepare the enzyme in the human N-linkedsugar chain synthesis system corresponding to the gene in a largeamount. The vector for use in the method for producing the enzyme of thepresent invention includes pBR322, pUC18, pET-3 and the like when thehost is Escherichia coli, and YEp13, YCp50, YEp51, YEp352GAP, pSH19,pREP1 and the like when the host is yeast. Furthermore, a promoter islinked to the upstream thereof so as to express the gene. The promoterused in the present invention may be any promoter, so long as it is asuitable promoter corresponding to the host used for expressing thegene. The host includes Escherichia coli (BL21, BL21(DE3), etc.), yeast(Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomiyces pombe,etc.), and the like.

The enzyme in the human N-linked sugar chain synthesis system obtainedby the above production method is useful as a therapeutic agent of CDGS,and a human N-linked sugar chain can be synthesized in vitro by usingthe enzyme. For example, in the case of α-mannosyltransferase encoded bythe human ALG 11 homologous gene, Man5GlcNAc2-pp-Dol can be synthesizedby using Man4GlcNAc2-pp-Dol and GDP-mannose as substrates.

Examples of the present invention are shown below; however,particularly, the present invention is not limited thereto.

EXAMPLE 1 Cloning of ALG11 Human Homolog FLJ21803

The gene was cloned by PCR using a human cDNA library. As the human cDNAlibrary, QUICK.Clone cDNA manufactured by CLONTECH was used. As primers,primers containing an NdeI site at the N-terminal and an SmaI site atthe C-terminal were prepared in advance, on the basis of sequencesregistered on the database so as to easily cleave a part encoding theprotein with restriction enzymes. Sequences of respective primers areshown below. 5′-TCCCCCGGGT TACTTAAATA ACTTTTCCAC AGATGATAGG AA-3′5′-GGGAATTCCA TATGGCGGCC GGCGAAAGGA GCTG-3′

PCR conditions are as follows: First stage: 94° C., 15 seconds Secondstage: 49° C., 30 seconds Third stage: 72° C., 3 minutes 30 Cycles

A DNA amplification fragment of about 1.5 kbp obtained under theconditions was inserted into a pCR2.1TOPO vector by using a TA cloningkit. The nucleotide sequence of the cloned gene was confirmed by asequence kit using the dideoxy method. The gene had the nucleotidesequence represented by SEQ ID NO: 1. Furthermore, SEQ ID NO: 1 alsoshows the amino acid sequence corresponding to the nucleotide sequenceof the gene. Additionally, the amino acid sequence of a proteincorresponding to the gene is represented by SEQ ID NO:2.

Transformation:

The FLJ21803 gene inserted into the pCR2.1TOPO vector was cleaved byNdeI-SmaI, and inserted into the NdeI-SmaI site of a vector for fissionyeast multicopy expression, pREP1, having a multicloning site betweenthe promoter nmt1 of fission yeast and the terminator thereof to therebyconstruct 21803/pREP1. The expression vector was transformed intoSchizosaccharomyces pombe gmd3 mutant strain of fission yeast.

Function of gmd3 Mutant Strain:

The temperature sensitivity of the resulting transformant was examined,which was an indicator of the presence or absence of N-linked sugarchain synthesis. The transformant and Schizosaccharomyces pombe JY746strain (wild type strain) and the gmd3 strain which were controls werecultured in an MM-leu medium having the following composition at 37° C.for 3 days to examine the temperature sensitivity.

As a result, it was confirmed that the transformant and the wild typestrain could grow even at 37° C. On the other hand, the gmd3 straincould not grow at 37° C.

Separately, the transformant which could grow at 37° C. was collected,and was grown in a low phosphoric acid medium and disrupted with glassbeads. The resulting product was used as a sample for electrophoresis onacrylamide gel. In the same manner, electrophoresis was carried out byusing a sample obtained from the gmd3 strain in which the ALG11 gene ofSaccharomyces cerevisiae was transformed, a sample of the gmd3 strain,and a sample of the JY746 strain (wild type strain). The results arecollectively shown in FIG. 1.

In the drawing, lanes 1 to 3 in the drawing show samples of thetransformant of Schizosaccharomyces pombe, lane 4 shows a sample of thegmd3 strain obtained by separate culture, lane 5 shows a sample obtainedfrom the gmd3 strain in which the ALG11 gene of Saccharomyces cerevisiaewas transformed, and lane 6 shows a sample obtained from the JY746strain (wild type strain).

In both of the transformant samples and the sample of the JY746 strain(wild type strain), bands corresponding to an acidic phosphatase havinga large molecular weight to which sugar chains were completely addedwere observed, whereas, in the sample of the gmd3 strain, only a band ofan acidic phosphatase having a molecular weight smaller than that of thebands, to which sugar chains were incompletely added, was observed.

Accordingly, these results clearly show that the human gene FLJ21803compliments the function in fission yeast.

EXAMPLE 2 Cloning of ALG8 Human Homolog MGC2840

Using a human cDNA library, the gene was cloned by PCR. As the humancDNA library, QUICK-Clone cDNA manufactured by CLONTECH was used. Theprimers were prepared on the basis of sequences registered on thedatabase. Sequences of the respective primers are shown below.5′-GGAATTCCAT ATGGCGGCGC TCACAATTG CCACGGGTAC TGGC-3′ 5′-TCCCCCGGGTCATTGTTTCT TTGTCTTGC CAATAGCAGA G-3′

PCR conditions are as follows: First stage: 94° C., 30 seconds Secondstage: 50° C., 30 seconds Third stage: 72° C., 2 minutes 30 Cycles

A DNA amplification fragment of about 1.5 kbp obtained under theconditions was inserted into a pCR2.1TOPO vector by using a TA cloningkit. The nucleotide sequence of the cloned gene was confirmed by asequence kit using the dideoxy method. The gene had the nucleotidesequence represented by SEQ ID NO:3. Furthermore, SEQ ID NO:4 shows theamino acid sequence corresponding to the gene.

Transformation:

The MGC2840 gene inserted into the pCR2.1TOPO vector was cleaved byEcoRI-NaeI, and then inserted into the EcoRI-PvuII site of a vector forexpression, YEp352GAO, having a part from the EcoRI region to the SalIregion in the multicloning site of pUC18 between a promoter GAPDH in theyeast glycolytic pathway and the terminator thereof. These expressionvectors were transformed into Saccharomyces cerevisiae alg8 wbp1 mutantstrain of budding yeast.

Recovery of Function of alg8 wbp1 Mutant Strain:

The temperature sensitivity of the resulting transformant was examined,which was an indicator of the presence or absence of N-linked sugarchain synthesis. The transformant and Saccharomyces cerevisiae W303-1Astrain (wild type strain) and the alg8 wbp1 mutant strain which werecontrols were cultured in an SD-ura medium having the followingcomposition at 30° C. for 5 days to examine the temperature sensitivity.

As a result, it was confirmed that the transformant and the wild typestrain could grow even at 30° C. On the other hand, the alg8 wbp1 mutantstrain could not grow at 30° C.

Separately, the transformant which could grow at 30° C. was collected,and was grown in a complete medium and disrupted with glass beads. Theresulting product was used as a sample for electrophoresis on acrylamidegel. In the same manner, a sample of the alg8 wbp1 strain and a sampleof the W303-1A strain (wild type strain) were subjected toelectrophoresis. The results are collectively shown in FIG. 2.

In the drawing, lanes 1 to 4 show samples obtained from the transformantof Saccharomyces cerevisiae, lane 5 shows a sample of the alg8 wbp1strain obtained by separate culture, and lane 6 shows a sample obtainedfrom the W303-1A strain (wild type strain). In both of the transformantsample and the sample of the W303-1A strain (wild type strain), bandscorresponding to a carboxypeptidase Y having a large molecular weight towhich sugar chains were completed added were observed, whereas, in thesample of the alg8 wbp1, only a band of a carboxypeptidase Y having amolecular weight smaller than that of the bands, to which sugar chainswere incompletely added, was observed.

Accordingly, these results clearly show that the human gene MGC2840compliments the function in budding yeast.

EXAMPLE 3 Cloning of ALG9 Human Homolog FLJ21845

Using a human cDNA library, the gene was cloned by PCR. As the humancDNA library, QUICK-Clone cDNA manufactured by CLONTECH was used. As theprimers, primers were prepared on the basis of sequences registered onthe database. Sequences of respective primers are shown below.5′-AACGTTAACA TGGCTAGTCG AGGGGCTCGG CAGCGCCTGA AGGGCAGC-3′ 5′-AACGTTAACCTAACCTCCAC TTTTCTTCCT GATTTGCTTT GCTTTCCG-3′

PCR conditions are as follows: First stage: 94° C., 30 seconds Secondstage: 50° C., 30 seconds Third stage: 72° C., 3 minutes 30 Cycles

A DNA amplification fragment of about 2 kbp obtained under theconditions was inserted into a pCR2.1TOPO vector by using a TA cloningkit. The nucleotide sequence of the cloned gene was confirmed by asequence kit using the dideoxy method. The gene had the nucleotidesequence represented by SEQ ID NO:5. Furthermore, SEQ ID NO:6 shows theamino acid sequence corresponding to the gene.

Transformation:

The FLJ21845 gene inserted into the pCR2.1TOPO vector was cleaved byEcoRI-DraI and then inserted into the EcoRI-PvuII site of a vector forexpression, YEp352GAP, having a part from the EcoRI region to the SalIregion in the multicloning site of pUC18 between a promoter GAPDH in theyeast glycolytic pathway and the terminator thereof. These expressionvectors were transformed into Saccharomyces cerevisiae alg9 wbp1 mutantstrain of budding yeast.

Recovery of Function of alg9 wbp1 Mutant Strain

The temperature sensitivity of the resulting transformant was examined,which was an indicator of the presence or absence of N-linked sugarchain synthesis. The transformant and Saccharomyces cerevisiae W303-1Astrain (wild type strain) and the alg9 wbp1 mutant strain which werecontrols were cultured in an SD-ura medium having the followingcomposition at 30° C. for 5 days to examine the temperature sensitivity.As a result, it was confirmed that the transformant and the wild typestrain could grow even at 30° C. On the other hand, the alg9 wbp1 mutantstrain could not grow at 30° C.

Separately, the transformant which could grow at 30° C. was collected,and was grown in a complete medium and disrupted with glass beads. Theresulting product was used as a sample for electrophoresis on acrylamidegel. In the same manner, a sample of the alg9 wbp1 strain and a sampleof the W303-1A strain (wild type strain) were subjected toelectrophoresis. The results are collectively shown in FIG. 3.

In the drawings, lanes 1 to 3 show samples obtained from thetransformant of Saccharomyces cerevisiae, lane 4 shows the sample of thealg9 wbp1 strain obtained by separate culture, lane 5 shows a sampleobtained from the W303-1A strain (wild type strain).

In both of the transformant sample and the sample of the W303-1A strain(wild type strain), bands corresponding to a carboxypeptidase Y having alarge molecular weight to which sugar chains were completely added wereobserved, whereas, in the sample of the alg9 wbp1, only a band of acarboxypeptidase Y having a molecular weight smaller than that of thebands, to which sugar chains were not added, was observed.

Accordingly, these results clearly show that the human gene FLJ21845compliments the function in budding yeast.

EXAMPLE 4 Cloning of ALG10 Human Homolog XM_(—)050190

Using a human cDNA, the gene was cloned by PCR. As the cDNA, humanstomach cDNA was used. As the primers, primers were prepared on thebasis of sequences registered on the database. Sequences of respectiveprimers are shown below. 5′-AAAAGGCCTA TGGCGCAGCT GGAAGGTTAC TATTTCTCGGCCGCCTTG-3′ 5′-TTTTCCGGAT TACCACATAA ACCTTTGAAT GTCCTGACTA TTTGGCCACT-3′

PCR conditions are as follows: First stage: 94° C., 30 seconds Secondstage: 50° C., 30 seconds Third stage: 72° C., 2 minutes 30 Cycles

A DNA amplification fragment of about 1.5 kbp obtained under theconditions was inserted into a pCR2.1TOPO vector by using a TA cloningkit. The nucleotide sequence of the cloned gene was confirmed by asequence kit using the dideoxy method. The gene had the nucleotidesequence represented by SEQ ID NO:7. Furthermore, SEQ ID NO:8 shows theamino acid sequence corresponding to the gene.

Transformation:

The M_(—)050190 gene inserted into the pCR2.1TOPO vector was cleaved byEcoRI-KpnI and then inserted into the EcoRI-KpnI site of a vector forexpression, YEp352GAO, having a part from the EcoRI region to the SalIregion in the multicloning site of pUC18 between a promoter GAPDH in theyeast glycolytic pathway and the terminator thereof. These expressionvectors were transformed into Saccharomyces cerevisiae alg10 wbp1 mutantstrain of budding yeast.

Recovery of Function of alg10 wbp1 Mutant Strain:

The temperature sensitivity of the resulting transformant was examined,which was an indicator of the presence or absence of N-linked sugarchain synthesis. The transformant and Saccharomyces cerevisiae W303-1Astrain (wild type strain) and the alg10 wbp1 mutant strain which werecontrols were cultured in an SD-ura medium having the followingcomposition at 30° C. for 5 days to examine the temperature sensitivity.

As a result, it was confirmed that the transformant and the wild typestrain could grow even at 30° C. On the other hand, the alg10 wbp1mutant strain could not grow at 30° C.

Separately, the transformant which could grow at 30° C. was collected,and was grown in a complete medium and disrupted with glass beads. Theresulting product was used as a sample for electrophoresis on acrylamidegel. In the same manner, a sample of the alg10 wbp1 strain and a sampleof the W303-1A strain (wild type strain) were subjected toelectrophoresis. The results are collectively shown in FIG. 4.

In the drawing, lanes 1 to 4 show samples obtained from the transformantof Saccharomyces cerevisiae, lane 5 shows the sample of the alg10 wbp1strain obtained by separate culture, and lane 6 shows a sample obtainedfrom the W303-1A strain (wild type strain).

In both of the transformant sample and the sample of the W303-1A strain(wild type strain), bands corresponding to a carboxypeptidase Y having alarge molecular weight to which sugar chains were completely added wereobserved, whereas, in the alg10 wbp1 sample, a band of acarboxypeptidase Y having a molecular weight smaller than that of thebands, to which sugar chains were not added, was observed.

Accordingly, these results clearly show that the human gene XM_(—)050190compliments the function in budding yeast.

EXAMPLE 5 Cloning of ALG12 Human Homolog MGC3136

Using a human cDNA library, the gene was cloned by PCR. As the humancDNA library, human tissue cDNA was used. As the primers, primers wereprepared on the basis of sequences registered on the database. Sequencesof respective primers are shown below. 5′-CGGAATTCAT GGCTGGAAAGGGGTCATCAG GCAGGCGG-3′ 5′-CGGAATTCTC AGGACGGCCG GGGGAGCCTC TCCAGAAGC-3′

PCR conditions are as follows: First stage: 94° C., 30 seconds Secondstage: 50° C., 30 seconds Third stage: 72° C., 3 minutes 30 Cycles

A DNA amplification fragment of about 1.5 kbp obtained under theconditions was inserted into a pCR2.1TOPO vector by using a TA cloningkit. The nucleotide sequence of the cloned gene was confirmed by asequence kit using the dideoxy method. The gene had the nucleotidesequence represented by SEQ ID NO:9. Furthermore, SEQ ID NO: 10 showsthe amino acid sequence of a protein corresponding to the gene.

Transformation:

The MGC3136 gene inserted into the pCR2.1TOPO vector was cleaved byEcoRI and then inserted into the EcoRI site of a expression forexpression, YEp352GAO, having a part from the EcoRI region to the SalIregion in the multicloning site of pUC18 between a promoter GAPDH in theyeast glycolytic pathway and the terminator thereof. These expressionvectors were transformed into Saccharomyces cerevisiae alg12 mutantstrain of budding yeast.

Recovery of Function of alg12 Mutant Strain:

The resulting transformant was collected, grown in a complete medium anddisrupted with glass beads. The resulting product was used as a samplefor electrophoresis using acrylamide gel. In the same manner, a sampleof the alg12 strain and a sample of the W303-1A strain (wild typestrain) were also subjected to electrophoresis. The results arecollectively shown in FIG. 5.

In the drawing, lanes 1 to 4 show samples obtained from the transformantof Saccharomyces cerevisiae, lane 5 shows the sample of the alg12 strainobtained by separate culture, and lane 6 shows a sample obtained fromthe W303-1A strain (wild type strain).

In the transformant sample and the sample of the W303-1A strain (wildtype strain), bands corresponding to a carboxypeptidase Y having a largemolecular weight to which sugar chains were completely added wereobserved, whereas, in the sample of the alg12 strain, only a band of acarboxypeptidase Y having a molecular weight smaller than that of thebands, to which sugar chains were not added, was observed.

Accordingly, these results clearly show that the human gene MGC3136compliments the function in budding yeast.

INDUSTRIAL APPLICABILITY

According to the present invention, the gene of the N-linked sugar chainsynthase in human endoplasmic reticulum has been found for the firsttime. It is known that the deletion or mutation of the gene of theN-linked sugar chain synthase in human endoplasmic reticulum causescongenital disorders of glycosylation syndrome (CDGS). Thus, the gene ofthe present invention is very useful for diagnosis and treatment forcongenital disorders of glycosylation syndrome (CDGS), and the like.

1. A human gene for synthesizing an enzyme catalyzing human N-linkedsugar chain synthesis, which is homologous with a gene of an enzymecatalyzing N-linked sugar chain synthesis in yeast endoplasmicreticulum, and is capable of complimenting the function of said gene fora deletion yeast strain of said gene.
 2. The human gene according toclaim 1, wherein the enzyme catalyzing human N-linked sugar chainsynthesis is a glycosyltransferase.
 3. A gene which encodes the aminoacid sequence represented by SEQ ID NO:2, 4, 6, 8 or 10 or a proteinwhich comprises an amino acid sequence in which one or more amino acidsin the amino acid sequence represented by SEQ ID NO:2, 4, 6, 8 or 10 aredeleted, substituted or added.
 4. The gene according to claim 3, whichcomprises the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7or
 9. 5. A method for diagnosing or treating human congenital disordersof glycosylation syndrome, which comprises using the gene encoding theamino acid sequence according to claim 3 or the gene represented by SEQID NO:1, 3, 5, 7 or
 9. 6. A recombinant vector which is integrated witha gene selected from the group consisting of: a human gene forsynthesizing an enzyme catalyzing human N-linked sugar chain synthesis,which is homologous with a gene of an enzyme catalyzing N-linked sugarchain synthesis in yeast endoplasmic reticulum, and is capable ofcomplimenting the function of said gene for a deletion yeast strain ofsaid gene; and a gene which encodes the amino acid sequence representedby SEQ ID NO:2, 4, 6, 8 or 10 or a protein which comprises an amino acidsequence in which one or more amino acids in the amino acid sequencerepresented by SEQ ID NO:2, 4, 6, 8 or 10 are deleted, substituted oradded.
 7. A transformant which is transformed by the recombinant vectoraccording to claim
 6. 8. A process for producing an enzyme catalyzinghuman N-linked sugar chain synthesis, which comprises culturing thetransformant according to claim 7 in a culture, and collecting theenzyme catalyzing human N-linked sugar chain synthesis from the culture.9. A method for synthesizing a human N-linked sugar chain, whichcomprises using the enzyme according to claim 8.